Chlamydiae are obligate intracellular microorganisms which parasitize eukaryotic cells and are ubiquitous throughout the animal kingdom. Members of the chlamydial genus are considered bacteria with a unique biphasic developmental cycle having distinct morphological and functional forms. This developmental growth cycle alternates between 1) intracellular life forms, of which two are currently recognized, a metabolically-active, replicating organism known as the reticulate body (RB) and a persistent, non-replicating organism known as the cryptic phase; and 2) an extracellular life form that is an infectious, metabolically-inactive form known as the elementary body (EB).
EBs are small (300-400 nm) infectious, spore-like forms which are metabolically inactive, non-replicating, and found most often in the acellular milieu. EBs are resistant to a variety of physical insults such as enzyme degradation, sonication and osmotic pressure. This physical stability is thought to be a result of extensive disulfide cross-linking of the cysteine-rich major outer membrane protein (MOMP) (Bavoil et al., Infection and Immunity, 44:479-485 (1984); Hackstadt et al., Journal of Bacteriology, 161:25-31 (1985); Hatch et al., Journal of Bacteriology, 165:379-385 (1986); Peeling et al., Infection and Immunity, 57:3338-3344 (1989); J. C. A. Bardwell, Molecular Microbiology, 14:199-205 (1994); and T. P. Hatch, Journal of Bacteriology, 178:1-5 (1993)). Under oxidizing conditions in the acellular milieu of the host, the outer membrane of EBs is relatively impermeable as well as resistant to inactivation. EBs are thus well suited to survive long enough outside of their hosts to be transmitted to a new host in the form of a droplet nuclei (Theunissen et al., Applied Environmental Microbiology, 59:2589-2593 (1993)) or a fomite (Fasley et al., The Journal of Infectious Diseases, 168:493-496 (1993)).
Once fully established, the Chlamydia are difficult to eradicate, with frequent relapse following antibiotic therapy. Evidence also indicates that the Chlamydia may become dormant and are then shed in quantities too few to reliably detect by culture.
C. pneumoniae is the most recent addition to the genus Chlamydiae and is well-accepted as a human pathogen that is difficult to eradicate by standard antibiotic therapy (Hammerschlag et al., Clin. Infect. Dis. 14:178-182 (1992)). C. pneumoniae is known to persist as a silent or mildly symptomatic pathogen, resulting in a chronic, persistent infection (Schacter, J., In: Baun AL, eg. Microbiology of Chlamydia, Boca Raton, Fla., CRC Press, 1988, pp. 153-165).
The current therapy for suspected/confirmed C. pneumoniae infection is with a short course (e.g., 2-3 weeks) of a single antibiotic. C. pneumoniae is susceptible in vitro to tetracyline, erythromycin, clarithromycin, and fluoroquinolones such as ofloxacin and sparfloxacin (Kuo et al., Antimicrob. Agents Chemother., 32:257-258 (1988); Welsh et al., Antimicrob. Agents Chemother., 36:291-294 (1992); Chirgwin et al., Antimicrob. Agents Chemother., 33:1634-1635 (1989); Hammerschlag et al., Antimicrob. Agents Chemother., 36:682-683 (1992); Hammerschlag et al., Antimicrob. Agents Chemother., 36:1573-1574); M. R. Hammerschlag, Antimicrob Agents Chemother., 38:1873-1878 (1994); M. R. Hammerschlag, Infect. Med., 64-71 (1994)). Despite this demonstration of in vitro susceptibility, C. pneumoniae infections may relapse following antibiotic therapy with these agents. In vitro studies on the persistence of Chlamydiae despite specific and appropriate antibiotic therapy have suggested that the presence of antibiotics promotes the formation of an intracellular, non-replicative state (Beatty et al., Microbiol. Rev. 58:686-699 (1994)), typically referred to as the latent or cryptic phase. This change can be thought of as a stringent response and is seen also with nutrient starvation and exposure to gamma-interferon. Removal of the stressful influence allows the organism to resume replication. Thus, in this way, the organism can escape current antibiotic therapy used in clinical practice.
In view of the chronic and persistent nature of chlamydial infections, there is a need for reliable, accurate methods for diagnosis of pathogenic infection as well as therapeutic approaches to manage the infection. Due to the highly infective nature of Chlamydia EBs and their ability to reinfect cells, there is also a need for antichlamydial therapy which totally eradicates this pathogen, including the non-replicating cryptic phase and the extracellular phase, as well as the replicating reticulate body (RB). Eradication of all phases of the organism from the host will prevent the long term sequelae of such chronic infections such as coronary artery atheromatosis and other idopathic inflammatory diseases, recognized now as being associated with C. pneumoniae infection.
Because they are intracellular parasites, micro-organisms such as the chlamydiae cannot be cultured without the use of animal or tissue cultures. Continuous cell lines routinely used to cultivate C. pneumoniae include HL, Hep2, HeLa, H-292, HuEVEC and McCoy cells; stocks can be obtained from a commercial supplier (e.g., Bartells), from the American Type Culture Collection (ATCC), or from The Washington Research Foundation in the case of HL cells.
The present invention pertains to methods for clearing biological material infected with Chlamydia to produce Chlamydia-free cell lines and animals, and to methods of maintaining biological material, e.g., cell lines and animals, such that they remain Chlamydia-free. According to the method, a biological material is cleared from Chlamydia infection by contacting the biological material with at least two agents, each of which is effective against a different phase of the chlamydial life cycle, until the biological material no longer tests positive for Chlamydia. The agents can be selected from the group consisting of a) agents effective against the cryptic phase of the chlamydial life cycle; b) agents effective against the elementary body phase of the chlamydial life cycle; and c) agents effective against the replicating phase of the chlamydial life cycle. In one embodiment, the agent effective against the elementary body phase is a disulfide reducing agent. In another embodiment, the agent effective against the cryptic phase is a nitroaromatic compound, such as nitroimidazoles, nitrofluans, analogs, derivatives and combinations thereof.
Biological material that has been cleared of Chlamydia infection, according to the methods of this invention, are also described. The biological material can be a continuous cell line such as HeLa-CF, HL-CF, H-292-CF, HuEVEC-CF and McCoy-CF; wherein xe2x80x9cCFxe2x80x9d is a shorthand annotation for xe2x80x9cChlamydia-freexe2x80x9d. Alternatively, the biological material can be an animal, such as a mouse, rabbit or other animal model, which is negative for Chlamydia.
The invention also pertains to methods of maintaining a Chlamydia-free status in animals and cell lines which have been cleared of Chlamydia infection by the methods of this invention, or have never been infected, such as their Chlamydia-free offspring or progeny. Cells or animals can be maintained as Chlamydia-free by maintaining them on antibiotics and/or treating their nutrients and environment to ensure that they are Chlamydia-free. Particularly, a source of nutrients to be administered to Chlamydia-free cells or animals can be treated to inactivate or remove any chlamydial elementary bodies therefrom. This can be accomplished by exposing the nutrients to gamma irradiation for a period of time and level of exposure adequate to inactivate the elementary bodies. In addition to, or alternatively, a source of nutrients can be passed through a filtration system to physically remove the chlamydial elementary bodies therefrom. Optionally, the source of nutrients can be first treated with a disulfide reducing agent, such as dithiothreitol, before the filtration step is performed. The filter should be of adequate size such that objects larger than 0.5 microns are prevented from passing through.
The invention further pertains to a diagnostic kit or pack comprising an assembly of materials selected from the group consisting of antibiotics, reagents, Chlamydia-free cell lines, and combinations thereof, or other materials that would be necessary to perform any of the methods described herein.